We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. In order to understand its mechanism of action, we constructed in collaboration with the laboratory of Yoshihiro Nakatani epitope-tagged CTCF molecules and used them to isolate complexes between CTCF and other proteins from HeLa cell extracts. Several proteins were identified, the most prominent of which was nucleophosmin, a protein that tends to concentrate in the nucleolus. Chromatin immunoprecipitation studies across the chicken beta-globin locus showed that nucleophosmin co-localizes with CTCF at the two boundary elements at either end of the locus. Furthermore, in cell lines carrying multiple copies of the 5' insulator, fluorescence in situ hybridization analysis revealed that the insulators were localized on the nucleosome surface. Localization depended on the presence of an intact CTCF binding site. These findings suggest a model for enhancer blocking activity in which nucleophosmin tethers the insulator to the nucleosome surface in a CTCF-dependent manner. This model has interesting parallels to one proposed for the gypsy insulator element in Drosophila, where quite different proteins are involved. The insulator also has the separate ability to protect against position effects reporter genes that are stably transfected into cell lines or animals, serving as a boundary against encroachment of condensed chromatin. We found that this protective ability is present in a ?core' element, 250 bp long, from within the 1.2 kb insulator, and that deletion of subregions within the core that contain the CTCF site do not affect activity. However four other subregions corresponding to binding sites for nuclear proteins are important for position effect protection (boundary function). We have now shown that one of these binding sites is specifically responsible for maintaining a high level of histone acetylation and methylation at sites associated with gene activation. This site binds a heterodimer of the proteins USF1and USF2, which in turn recruit a variety of histone modifying enzymes to the site, including known acetylases and methylases. These results are consistent with a model we have proposed in which barrier function is connected with multiple histone modifications in the neighborhood of the insulator. We have also shown that other sites in the core region are responsible for inhibiting DNA methylation at a nearby locus. The globin insulator appears to serve in vivo as a barrier against encroachment of an upstream region of condensed chromatin. We have also collaborated with the laboratory of Hannah Gould to examine the relationship between chromatin structure and class switch recombination in the human immunoglobulin heavy chain locus. Earlier opinion has held that the choice of isotype was governed by the selective opening of chromatin at a single germ line gene. We showed by analysis of single B-cells that single cells contain transcripts from more than one germ line gene, implying that more than one gene can exist in an open chromatin structure in a given cell. We have also collaborated with the laboratory of Marisa Bartolomei in a study of the imprinted Igf2/H19 locus in mouse. Our earlier work had shown that imprinting depends on the methylation in the paternal allele of CTCF binding sequences within the imprinting control region within the locus. The new data were obtained in mice which had mutations in the CTCF sites that prevented methlyation but not CTCF binding. Maternal inheritance of the mutation left Igf2 and H19 patterns of expression intact, while paternal inheritance allowed insulation and consequent Igf2 suppression on that allele and relief of repression of H19, normally observed in wild type cells. The results provide further information on the generation of the imprinting marks.